Peptidome profiling for the immunological stratification in sepsis: a proof of concept study

Sepsis has been called the graveyard of pharmaceutical companies due to the numerous failed clinical trials. The lack of tools to monitor the immunological status in sepsis constrains the development of therapies. Here, we evaluated a test based on whole plasma peptidome acquired by MALDI-TOF-mass spectrometer and machine-learning algorithms to discriminate two lipopolysaccharide-(LPS) induced murine models emulating the pro- and anti-inflammatory/immunosuppression environments that can be found during sepsis. The LPS group was inoculated with a single high dose of LPS and the IS group was subjected to increasing doses of LPS, to induce proinflammatory and anti-inflammatory/immunosuppression profiles respectively. The LPS group showed leukopenia and higher levels of cytokines and tissue damage markers, and the IS group showed neutrophilia, lymphopenia and decreased humoral response. Principal component analysis of the plasma peptidomes formed discrete clusters that mostly coincided with the experimental groups. In addition, machine-learning algorithms discriminated the different experimental groups with a sensitivity of 95.7% and specificity of 90.9%. Data reveal the potential of plasma fingerprints analysis by MALDI-TOF-mass spectrometry as a simple, speedy and readily transferrable method for sepsis patient stratification that would contribute to therapeutic decision-making based on their immunological status.

Supplementary figure S1. Inoculation and sample collection schemes. Inoculation schemes of the proinflammatory (LPS) group, the anti-inflammatory/immunosuppression (IS) group and the basal control (CTL) group. Plasma were collected in heparinized tubes at the indicated time points through submandibular bleeding. i.p.: intraperitoneally.
Supplementary figure S2. Peripheral blood leukocytes count. BALB/c mice were inoculated with one LPS dose (proinflammatory state; LPS group) (a), or with successive and increasing LPS doses (immunosuppression state; IS group) (b). Inoculation and blood collection schemes are detailed in the Suppl. Fig. S1. Blood was collected after 1.5 h (LPS group) or 24h after the last LPS dose LPS (IS group). A control group (CTL) was inoculated with vehicle (saline solution) and the plasma was collected at the same time points. The samples were analyzed with a Coulter hematology analyzer. Results are expressed as the mean ± SEM; n= 6 to 7 per group. Data are representative of two independent experiments. ****P<0.0001 compared to the same cell type in the CTL group. One-way ANOVA and Tukey's multiple comparisons test. Ly: lymphocytes; Mo: monocytes; Gra: granulocytes.

Supplementary figure S3.
Pro and anti-inflammatory cytokines levels in plasma. BALB/c mice were inoculated with one LPS dose (pro-inflammatory state; LPS group) (a, b), or with successive and increasing LPS doses (immunosuppression state; IS group) (c). At different times after the last LPS challenge plasma were collected, and the cytokines levels were evaluated through an ELISA. (a, b) TNF-α, IL-6, IL-10 and TGF-β were evaluated 1.5h post LPS; IL-12 and IFN-γ at the 6h post LPS challenge. (c) Cytokines TNF-α, IL-6, IL-10 and TGF-β were evaluated 24h after the last LPS dose. A control group (CTL) was inoculated with vehicle (saline solution) and the plasma was collected at the same time points. Results are expressed as the mean ± SEM; n= 6 to 7 per group. Data are representative of two independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with CTL mice at the same time; Mann-Whitney test.
Supplementary figure S4. Tissue damage indicators in plasma. BALB/c mice were inoculated with one LPS dose (proinflammatory state; LPS group) (a, b, c), or with successive and increasing LPS doses (antiinflammatory/immunosuppression state; IS group) (d, e, f). After 6 h (LPS group) or 24 h (IS group) after the last LPS administration, plasma were collected and enzyme levels were evaluated. A control group (CTL) was inoculated with vehicle (saline solution) and the plasma were collected at the same time points. AST: aspartate transaminase; ALT: alanine transaminase; Cre: creatinine. Results are expressed as the mean ± SEM; n= 5 to 7 per group. Data are representative of two independent experiments. *P<0.05, **P<0.01, ***P<0.001 compared with CTL mice at the same time; Student's t-test.

Supplementary figure S5.
Lymphocyte and myeloid cell populations in spleen and humoral immune response during the immunosuppression phase. BALB/c mice were inoculated with successive and increasing LPS doses (immunosuppression state; IS group). A control group (CTL) was inoculated with vehicle (saline solution). 24h after the last LPS dose or vehicle, the spleen were removed and the cellular suspension was evaluated by flow cytometry. (a) Total numbers of B lymphocytes (B Ly), CD4 and CD8 T lymphocytes (Ly CD4; Ly CD8) and neutrophils (Neu; CD11b/Ly6G) from the IS and CTL spleen were evaluated. Ly and myeloid gates were defined by features of forward and side scatter. (b) PDL-1 expression on splenic CD11b myeloid cells of IS and CTL mice was evaluated. Results are expressed as the mean ± SEM; n= 6 to 8 per group. Data are representative of two independent experiments. **P<0.01, ****P<0.0001 compared with to the same cell type in the CTL group; Student's t-test. (c) BALB/c mice from IS were immunized with sheep red blood cells (SRBCs; 5x10 8 /mouse, 0.1 ml i.p.) 24 h after the last LPS dose. CTL mice were immunized with the same antigen. Seven days after the immunization, the mice were bled and the serum was collected. The anti-SRBC antibody titer was evaluated by hemagglutination assay. Results are expressed as the mean ± SEM; n= 8 per group. Data are representative of two independent experiments. ****P<0.0001 compared with the CTL group; Student's t-test.

Supplementary figure S6.
Representative average mass spectra of each experimental group. The y-axis represents the calibrated intensity of each detected peaks, which are represented in the x-axis. Each image represents the average of 2 mass spectra acquired in duplicate.